Mycobacterium tuberculosis latent infection in healthcare students: systematic review of prevalence

ABSTRACT Objective: The aim of this study was to synthesize the evidence on the prevalence of latent Mycobacterium tuberculosis infection (LTBI) among undergraduate health care students. Methods: A systematic review of prevalence with meta-analysis was conducted. Prospective and retrospective cohorts and cross-sectional studies involving probable exposure to M. tuberculosis during undergraduate education, along with the tuberculin skin test (TST) or interferon-γ release assay (IGRA) for investigation of latent tuberculosis were searched. Searches were conducted in MEDLINE, CINAHL, EMBASE, LILACS, Scopus, and Web of Science databases. Independent reviewers were responsible for the selection and inclusion of studies. Data were extracted, critically appraised, and synthesized using the JBI approach. PRISMA was used to report the study. Results: Twenty-two studies were analyzed. The overall prevalence in healthcare undergraduate students was 12.53%. Conclusion: The prevalence of LTBI in undergraduate health students was high for such a highly educated population. Screening with TST and/or IGRA and chemoprophylaxis, when necessary, should be provided to undergraduate health students when in contact with respiratory symptomatic patients.


INTRODUCTION
Tuberculosis is an infectious disease, caused by the bacterium Mycobacterium tuberculosis, ranked among the top ten causes of death worldwide.Moreover, it is expected to be the second leading cause of death from a single infectious agent, until the COVID-19 pandemic (1) .Although tuberculosis is treatable, about 10 million new cases were reported globally in 2021, and almost 1.6 million people died from the disease, even with a reduction in notifications during the pandemic scenario (1) .
Tuberculosis primarily affects the lungs, and its contagion occurs through inhalation of droplets released by patients with active infection (2) .The risk of infection is directly related to the intensity and time of exposure to a bacilliferous person (3) .When the bacteria are inhaled, the immune system develops defense mechanisms through macrophages, producing granulomas.In most people (about 90%), the infectious process is maintained with bacterial replication in equilibrium and latent infection, called latent M. tuberculosis infection or latent tuberculosis infection (LTBI) (2) .
While infectious expression in LTBI is not induced, the body continues to generate an ongoing immune response to tuberculosis, with a 10% probability of progression to active tuberculosis (4) .Therefore, the World Health Organization (WHO) ranks the detection and prevention of LTBI as a crucial strategic component to prevent active tuberculosis (4) .
Global strategies and guidelines outline the importance of LTBI screening and treatment associated with early diagnostic testing to prevent new cases (5) .As no direct diagnostic methods of LTBI currently exist, T-cell response diagnostics are used, which are tuberculin skin test (TST) and interferon-gamma release assays (IGRA) (4) .
The TST and IGRA are the most relevant tests for identifying LTBI because, although there is no diagnosis for active tuberculosis, they indicate whether the individual has had contact with M. tuberculosis (6) .The TST induces a hypersensitivity reaction via an intradermal injection of a purified protein derivative (PPD), which contains antigens from M. tuberculosis, nontuberculous mycobacteria, and Mycobacterium bovis Bacillus Calmette-Guerin (BCG).IGRA are in vitro blood tests that measure the amount of interferon-gamma (IFN-γ) produced by CD4+ T lymphocytes or the number of IFN-γ-producing T cells following stimulation with specific ESAT-6 and CFP-10 antigens of M. tuberculosis (7) , showing more specificity than TST (3,8) .
Healthcare workers are among the most vulnerable groups to LTBI, which also include the following populations: those living on the street, deprived of liberty, and living with HIV, as well as migrants and indigenous peoples (1,9,10) .Health care professionals who are exposed to patients with the active form of tuberculosis are at greater risk of acquiring and, consequently, transmitting the bacillus (11) .
Undergraduate health students involved in clinical practice are also exposed to occupational risks similar to healthcare workers (3) .Nursing students at a Brazilian public university showed an estimated 36% prevalence of LTBI (3) .Exposure of undergraduate health students to M. tuberculosis in high-prevalence settings is almost inevitable, as their training must take place in healthcare facilities with a high risk of infection (12) .
Therefore, the aim of this study was to synthesize the evidence on the prevalence of latent tuberculosis infection among undergraduate health students.The research question that guided the study was: What is the prevalence of latent tuberculosis infection among undergraduate health students?

METHODS
This is a systematic review of prevalence, developed according to the Joanna Briggs Institute ( JBI) recommendations (13) .The protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) under the identification number CRD42020190716.The final report was constructed according to the PRISMA recommendations (14) .
This review included observational studies published in English, Portuguese, or Spanish, involving probable exposure to M. tuberculosis during undergraduate health education (academic activities and interaction with other students), along with the use of TST and/or IGRA to diagnose latent tuberculosis.
Studies involving the participation of students working in health services concurrently with those diagnosed with tuberculosis before joining the undergraduate course were excluded.

InformatIon SourceS and Search StrategIeS
The search for the articles was performed in the CINAHL, EMBASE, LILACS, and MEDLINE via PubMed, Scopus, and Web of Science databases.The first search took place in September 2021 and the last search update took place in November 2022.The search strategies are available at the supplementary material Chart 1.

Study SelectIon
Subsequent to the searches on the information sources, duplicates were removed.Titles and abstracts were independently reviewed by three reviewers based on the eligibility criteria, and then the entire texts were analyzed by the same group of reviewers.Mendeley software was used to manage the references.All studies excluded in the text-screening phase were characterized regarding the reasons for exclusion.

data extractIon
Data such as citation, study design, methods, country, subject, setting, year of data collection, participant characteristics, method of outcome measurement, and LTBI prevalence were independently extracted by two reviewers using a data extraction form created exclusively for this study.This form was based on the Joanna Briggs Institute ( JBI) template for systematic reviews of prevalence and incidence (13) .Disagreements were discussed to reach consensus, but when this was not possible, a third, more experienced reviewer was activated for final decision.

rISk of BIaS and certaInty of evIdence aSSeSSment
The GRADE -Grading of Recommendations Assessment, Development, and Evaluation (15) was used to analyze the quality of the studies (16) .Methodological limitations in the study design and execution of methodological steps were assessed by means of the Joanna Briggs Institute checklists used for analytical crosssectional studies (17) , prevalence studies (18) , and cohort studies (18) .Disagreements were resolved by consensus.
Heterogeneity was assessed using the I 2 and X 2 tests.A value greater than 50% was considered an indicator of substantial heterogeneity among studies.The level of statistical significance was fixed at 0.05 (p > 0.05).Indirectness and imprecision were evaluated with caution.Publication bias was assessed by means of funnel plots (19) , using the R x64 4.0.0 software, via RStudio Desktop.

data analySIS and SyntheSIS
Qualitative synthesis was performed in narrative and tabular form.Quantitative synthesis was performed via OpenMeta[Analyst] software, using the logit transformed proportion method in the random model with confidence interval established at 95%.

Study SelectIon
A total of 121 articles were found during the database searches.After removing 44 duplicates, 77 articles had their title and abstract screened.Twenty-five articles had their fulltext screened, but only 22 studies were included in the sample.The reasons for exclusion of studies at the full-text screening phase was language (n = 1) and different outcome (n = 2).The PRISMA flow diagram is shown in Figure 1.

rISk of BIaS In the StudIeS
The results of using the Joanna Briggs Institute checklists for cohort studies and analytical cross-sectional studies are available at the supplementary material tables 2 and 3 (https://osf.io/download/93ucz/).An important risk of bias was neglecting the confounding variables in both cohort studies and analytical cross-sectional studies.History of tuberculosis diagnosis, of direct contact with tuberculosis patients, of working in health care, and of presence of immunosuppressive condition or treatment and BCG scar are the main confounding variables, and few studies (A5 (24) , A8 (27) , A10 (29) , and A22 (41) ) accounted for them adequately.

QualItatIve SyntheSIS
The prevalence of LTBI among undergraduate health students ranged from 1.1% (95% CI 0.7-1.6%) in a study conducted in Italy (24) to 50.23% (95% CI 45.43-55.02%) in another study from India (29) .Table 1 presents the main results of the individual studies, including sample size, frequency of LTBI diagnoses, prevalence expressed as a percentage, the country in which the study was conducted, and whether that country has high tuberculosis, tuberculosis/HIV, and/or MDR/RR-tuberculosis burden, according to the World Health Organization (42) .
Nine studies have shown the prevalence of LTBI from both among undergraduate health students in the pre-clinical phase (before entering health care facilities) or undergraduate students from other nonclinical areas (those that do not involve entering health care facilities) and among undergraduate health students in the clinical phase (inside health care facilities).Data of sample sizes, frequency of LTBI diagnoses, and prevalence expressed as percentages in the pre-clinical, pre-non-clinical, and clinical phases, and whether the country in which the study was conducted has high tuberculosis, tuberculosis/HIV, and/or MDR/RR-tuberculosis burden are available at the supplementary material table 4 (https://osf.io/download/93ucz/).The presence of a BCG vaccine scar was evaluated in 3 studies.One of these studies (35) was conducted at the University of Putra Medical School in Malaysia, where 85.3% and 97.3% of 1st year and 5th year students had BCG vaccine scarring, respectively.Another study, conducted at the Universidade Autônoma de Querétaro in Mexico (23) , found that 100% of nursing students had a BCG scar.A study conducted at Addis Ababa University, in Ethiopia (32) , identified that 44.9% of medical students had BCG vaccine scar.
Two studies evaluated the likelihood of tuberculin skin test (TST) positive detection in students with BCG vaccine scars, especially those who received the booster dose of the vaccine, when compared to those who were not vaccinated.Both comparisons showed no statistically significant differences (26,28) .Another study found no statistically significant association between TST positivity and age, sex, or BCG vaccination in medical and nursing students from Italy (21) .Two other studiesone conducted with medical students in Brazil (36) and another with nursing students in India (29) -showed that the BCG scar was statistically significantly associated with positive TST result.Research conducted in Italy, with medical students, showed no statistically significant association between the presence of a BCG vaccine scar, or originating from countries with a high tuberculosis burden and a positive TST result (26) .Another study, whose participants were undergraduate and graduate health students in Italy, found that 17.0% of graduate medical students received BCG vaccination compared to 0.24% of undergraduate students (20) .Four studies showed reduced diagnoses of LTBI when, in addition to TST, confirmation was performed with the IGRA via Quantiferon-Tuberculosis test (QFT) (20,21,24,32) .

QuantItatIve SyntheSeS
Figure 2 presents the forest plot that summarizes the overall prevalence of LTBI among undergraduate health students.Other forest plots of the additional sub-group analysis are available at the supplementary material figures 1-4 (https://osf.io/download/93ucz/).

QualIty of the evIdence
The certainty of the body of evidence was very low for the main outcome, downgraded from high due to risk of bias, heterogeneity, and publication bias.The certainty of the body of evidence was low for all subgroup analysis due to risk of bias, heterogeneity, and publication bias.
The funnel plots (19) of the main meta-analysis and of the additional sub-group analysis are available at the supplementary material figures 5-9 (https://osf.io/download/93ucz/).All tests indicated asymmetries typical of publication bias, although all meta-analyses, except the one with all the included studies, showed small number of studies, and the low proportion of the outcomes may have overestimated the asymmetries (43) .

DISCUSSION
This systematic review showed that, similar to health professionals, students in health professions are also at significant risk of having been diagnosed with LTBI.In a population from www.scielo.br/reeuspMycobacterium tuberculosis latent infection in healthcare students: systematic review of prevalence Rev Esc Enferm USP • 2024;58:e20230238 which is expected knowledge regarding tuberculosis transmission and prevention as well as access to personal protective equipment (PPE), the overall prevalence in healthcare undergraduate students was 12.53%, while the overall prevalence across all populations ranged from 17.4% to 24.2% (44) .The main limitation of this systematic review is the language restriction.
In order to minimize the risks inherent to occupational exposure to which health students are subjected, the importance of educational programs on the proper use of PPE is relevant, as its proper use effectively prevents transmission by contact and droplets (45)(46)(47) .PPE is put on and taken off incorrectly by healthcare professionals very often (48) .Therefore, it is suggested that a significant number of infections could be avoided by professionals and students using individual protective measures.Education and improved awareness of the risks of acquiring tuberculosis are required to reduce the possibility of infection, with tutors expected to instruct their students on the correct management practices and use of PPE (49) .
A study conducted in India demonstrated that nursing and medical students are 2.54 times more likely to suffer from ILTB than non-healthcare undergraduate students, and there is a statistically significant increase in positivity for TST, as the duration of exposure to clinical practices increases (39) .During the early (pre-clinical) years, minimal exposure to clinical facilities occurs.However, students in later undergraduate terms are at higher risk for LTBI, as they are expected to spend more time in hospital settings to experience closer contact with patients (35) , increasing their risk of exposure to tuberculosis cases.
Regions of high tuberculosis incidence and prevalence have a high possibility of contact with tuberculosis patients, although it is more difficult to determine whether the exposure was actually occupational or communal.In countries with a high burden of tuberculosis, such as India, inadequate or no screening of outpatients with proven or suspected tuberculosis, inadequate ventilation, and overcrowding are observed in settings in which care is provided (29) .In this setting, healthcare professionals and undergraduate students provide care while maintaining close contact with patients with infectious tuberculosis (29) .
A study conducted in Brazil (40) showed that the time allocated to practical teaching of tuberculosis management ranged between 10 and 20 hours and occurred mostly in Primary Health Care services.Further studies in different countries would help determine in which health services undergraduate health students are most vulnerable in clinical practice.
Biological and social factors have a direct impact on the vulnerability to tuberculosis, such as malnutrition, age group, HIV infection, unhealthy housing, high population density, difficult access to health services, inadequate working conditions, among others (50) .In addition to these factors, this study, as well as others (21,35) , has shown that exposure during clinical practice in undergraduate health courses also increases the risk of exposure to M. tuberculosis, and consequently, LTBI.Thus, it is recommended that TST be conducted among undergraduate health students, both at baseline and throughout the course, to screen for LTBI as part of a tuberculosis screening program, which would include periodic clinical assessment.
The primary limitation of this review is the comprehensiveness of the searches.The inclusion criteria were restricted to the English, Portuguese, and Spanish languages, so relevant studies published in other languages may exist and were not included.Furthermore, despite consulting five of the most relevant health databases, relevant documents published in journals indexed in other databases may exist and were not included.Future research investigating the effectiveness of annual screening with TST and/or IGRA in health students, as well as chemoprophylaxis in cases of LTBI in this population, should be conducted.

CONCLUSION
The prevalence of LTBI in undergraduate health students was 12.53%, a significant and elevated proportion for a highly educated population, which is expected to have access to and adequate instruction in the use of PPE.This systematic review has contributed to evidence that, in addition to professionals, health care students are also a vulnerable group to LTBI.Annual screening for tuberculosis, including the TST and/or IGRA, in undergraduate health students from the beginning of their courses, can both facilitate the early diagnosis of LTBI, anticipate chemoprophylaxis, and prevent the manifestation of tuberculosis in this population.

Figure 2 -
Figure 2 -Forest plot of overall LTBI prevalence among undergraduate health students.
Chart 2 -Main characteristics of the studies.

Table 1 -
Prevalence of latent Mycobacterium tuberculosis infection among undergraduate health students identified in the individual studies.